Y Receptor-mediated Induction of CD63 Transcripts, a Tetraspanin Determined To Be Necessary for Differentiation of the Intestinal Epithelial Cell Line, hBRIE 380i Cells

Abstract

Peptide YY (PYY) and neuropeptide Y (NPY) are peptides that coordinate intestinal activities in response to luminal and neuronal signals. In this study, using the rat hybrid small intestinal epithelial cell line, hBRIE 380i cells, we demonstrated that PYY-and NPY-induced rearrangement of actin filaments may be in part through a Y1␣ and/or a nonneuronal Y2 receptor, which were cloned from both the intestinal mucosa and the hBRIE 380i cells. A number of PYY/NPY-responsive genes were also identified by subtractive hybridization of the hBRIE 380i cells in the presence or absence of a 6-h treatment with PYY. Several of these genes coded for proteins associated with the cell cytoskeleton or extracellular matrix. One of these proteins was the transmembrane-4 superfamily protein CD63, previously shown to associate with ␤ 1 -integrin and implicated in cell adhesion. CD63 immunoreactivity, using antibody to the extracellular domain, was highest in the differentiated cell clusters of the hBRIE 380i cells. The hBRIE 380i cells transfected with antisense CD63 cDNA lost these differentiated clusters. These studies suggest a new role for NPY and PYY in modulating differentiation through cytoskeletal associated proteins.Regulatory peptides common to the intestine and nerves (neuro-gut regulatory peptides) are unique chemical mediators released from receptosecretory and/or neurosecretory cells having multiple biological actions. These peptides can act as neurotransmitters, neurocrine, endocrine, or paracrine agents and share a common function in the coordination of intestinal activities in response to luminal signals or signals originating from peripheral tissues. Despite the range of diversity of their biological effects, there is one recurring biological observation that seems to link many of these peptides, their ability to act as factors affecting growth or differentiation of the intestinal mucosa. Differentiation and regulation of growth of complex tissues, such as the intestine, are not only determined by growth factors but also by intracellular signals generated by the interactions between cells and their extracellular matrix (ECM) 1 (1).In intestinal epithelial cells, part of the ECM occurs in the form of a basement membrane that provides these cells with positional information and signals that initiate the organization of intracellular structure and cellular behavior. A factor probably important for the positional cues for differentiation in the intestinal mucosa is the potential link between the ECM of the lamina propria and the process of cellular migration from crypt to villus. Although over the past years there have been studies on the growth effects for specific peptides such as cholecystokinin (2), bombesin (3), vasopressin, gastrin (4, 5), and peptide YY (PYY) (6), to name a few, the contribution of these peptides to intestinal differentiation remains relatively unexplored. Such investigations were limited in the past primarily due to the lack of an appropriate cell culture model that mimicked some of the critica...

Peptide YY and Neuropeptide Y Induce Villin Expression, Reduce Adhesion, and Enhance Migration in Small Intestinal Cells through the Regulation of CD63, Matrix Metalloproteinase-3, and Cdc42 Activity

“…PYY and NPY Reduce CD63 mRNA Expression in Pre-confluent Cells and Reduce Cell Adherence in a Time-and Dosedependent Fashion-PYY/NPY induce I-FABP and CD63 in post-confluent fully differentiated hBRIE 380i cells (31,32). The expression levels of CD63 affect those of I-FABP.…”

“…Antisense CD63 experiments utilized the plasmid construct described previously (32), which contained the full CD63 open reading frame inserted into the expression vector in the antisense orientation.…”

“…PYY and NPY Induce Villin Expression through a Pathway Involving MMP-3 Activity-Previously, we observed that modulation of cell-substratum interaction mediated by CD63 could be a pathway of PYY/NPY-induced expression of I-FABP (a late marker of cell differentiation) (32). To explore the possibility that PYY/NPY-induced cell migration also has an effect on the expression of villin (an early marker of cell differentiation) in hBRIE 380i cells, we first determined whether alteration of MMP-3 levels affects villin expression.…”

“…We previously reported that PYY and NPY induce the expression of a late marker of intestinal cell differentiation, intestinal fatty acid-binding protein (I-FABP) (31), and the tetraspanin CD63, a putative integrin-associated adhesive molecule (32), in non-migrating differentiated small intestinal hBRIE 380i cells (33). Reduction of CD63 transcript levels delays the expression of I-FABP (32).…”

“…We previously reported that PYY and NPY induce the expression of a late marker of intestinal cell differentiation, intestinal fatty acid-binding protein (I-FABP) (31), and the tetraspanin CD63, a putative integrin-associated adhesive molecule (32), in non-migrating differentiated small intestinal hBRIE 380i cells (33). Reduction of CD63 transcript levels delays the expression of I-FABP (32). Because CD63 has been implicated as necessary for ␤ integrinassociated cell adhesion and motility in breast carcinoma cells (34), we investigated whether PYY and NPY regulation of CD63 affects the expression of differentiation-dependent proteins such as villin before the cells develop a fully differentiated phenotype and whether this process involves modulation of cell adhesion and/or migration.…”